Okra is a very important vegetable crop of tropical countries. Diseases caused by virus such as Yellow Vein Mosaic Virus (YVMV) and Enation Leaf Curl Virus (EnLCuV) are the major problems associated with Okra cultivation. Development of virus resistance through conventional breeding is difficult due to lack of sources for resistance in the available germplasm.
Development of virus resistance by incorporating virus resistant genes through genetic engineering approach is the most promising alternative. For successful genetic transformation, it is desirable to have highly efficient regeneration methodology. Somatic embryogenesis is an effective tool in genetic transformation for the successful development of a large number of transgenic plants. Okra is known as a highly recalcitrant crop for tissue culture and regeneration. The available regeneration methodologies are based on direct shoot organogenesis from explants which are not desirable for genetic transformation as this may generate high frequency of chimeras and could also be extremely laborious.
U.S. Pat. No. 8,067,673 discloses a method for the regeneration and transformation of Okra and other Abelmoschus species into a whole plant using Agrobacterium-mediated gene transfer or particle bombardment methods. Transgenic insect resistant Okra plants were generated either by marker based or marker-free systems.
U.S. Pat. No. 8,168,748 discloses transgenic plant comprising transforming plant cells using viruses and Agrobacterium, physicochemical methods such as electroporation, polyethylene glycol, biolistic or particle bombardment, micro injection, floral dip method and others. The transformed plant cells comprising the isolated nucleic acids result in increased resistance from viruses such as a geminivirus, a nanovirus and combinations thereof.
U.S. Pat. No. 8,697,445 B2 discloses regeneration and genetic transformation of Okra with the use of meristematic cells of plumule tip through Agrobacterium-mediated transformation or bombarding explants of development of transgenic plants with insect resistance.
Patent application WO2011087854 discloses a method for producing a plant that has increased resistance to a single stranded DNA (ssDNA) virus of the geminivirus family comprising transforming a polynucleotide though an expression vector into said plant.
Another patent application US20020083491 discloses a method of using geminivirus vectors for silencing of one or more endogenous genes in treated plants.
The study of the prior art shows that reported methods on regeneration and genetic transformation of Okra were based on direct organogenesis of shoots from shoot-tips, cotyledonary node, plumule tip and immature embryo-based explants.
The available regeneration methodologies are based on direct shoot organogenesis from explants which are not desirable for genetic transformation as this may generate high frequency of chimeras and could also be extremely laborious. Somatic embryogenesis is an effective tool in genetic transformation for the successful development of a large number of transgenic plants. So far Okra is considered as a highly recalcitrant crop for tissue culture and regeneration through Somatic Embryogenesis.
The current invention solves the existing problem by providing methods for regeneration and genetic transformation of Okra through somatic embryogenesis in semisolid culture. This invention also provides a method for the development of transgenic Okra plants with virus-resistant gene constructs.